An important goal of in-vivo microscopy is diffraction limited imaging from layers deep within biological specimens. In practice, diffraction limited resolution is often not achievable due to distortions of the wave-front introduced by refractive index inhomogeneities in the specimen.
An analogue problem is present in astronomy where wave-front distortions may be caused by refractive index variations due to turbulences in the atmosphere. These distortions are routinely corrected using adaptive optics (see F. Merkle et al. “Successful Tests of Adaptive Optics” in “ESO messenger”, vol. 58, 1989, pages 1-4). Adaptive wave-front correction can be obtained by determining the distortion e.g. with the help of a bright star or an artificial reference such as a “laser guide-star”. The wave-front distortion is directly measured by subjecting light from the reference star e.g. to a Shack-Hartmann wave-front sensor (B. Platt et al. “Lenticular Hartmann Screen” in “Optical Sciences Center Newsletter”, vol. 5, 1971, p. 15). In the Shack-Hartmann wave-front sensor, the wave-front to be measured is directed to a regular array of microlenses. In the focal plane of these lenses, a CCD camera detects the light distribution of all lenses passed by the wave-front. The wave-front is reconstructed numerically from this distribution of light portions.
In microscopy, the adaptation technique known from infrared astronomy generally cannot be applied since neither natural nor artificial “guide stars” are available in a specimen like e.g. a biological specimen. The only situation where wavefront measuring techniques from astronomy can be directly applied is when the light comes primarily from the focal plane. This is the case in the eye, where a single tissue layer (the retina) reflects or backscatters most of the light, which can than be used to determine the distortion caused by lens and cornea (see J. F. Bille et al. “Imaging of retina by scanning laser tomography” in “Proc. SPIE”, vol. 1161, 1989, p. 417-425; J. Z. Liang et al. “Supernormal vision and high-resolution retina imaging through adapted optics” in “J. Opt. Soc. Am. A”, vol. 14 (11), 1997, p. 2884-2892).
Generally, biological or synthetical specimens have an inhomogeneous structure with different refractive indices in different structures. As an example, in a biological specimen a blood vessel may lie between a focal region of interest and a surface of the specimen causing an essential distortion of the light to be detected. For these less favourable biological specimens, wave-front measuring techniques from astronomy cannot be directly applied. Therefore, techniques have been proposed, which use the fact that one has considerable control of and information about the wave-front of the light source (e.g. scanning laser) for illuminating the specimen.
One conventional approach for wave-front correction in multiphoton or confocal microscopy is an iterative wave-front optimization using a search algorithm based on trial distortions of the incident wave-front. As an example, O. Albert et al. (“Smart microscope: an adaptive optics learning system for aberration correction in multi-photon confocal microscopy” in “Optics Letters”, vol. 25, 2000, p. 52-54) propose 5 an illumination of the specimen with a computer-controlled deformable mirror in conjunction with a learning algorithm to compensate for the static off axis aberrations. This approach uses the fact that the amount of fluorescence generated in the focal plane of the specimen strongly depends on the quality of the focus. Similar iterative wave-front optimization procedures are described by L. Sherman et al. (“Adaptive correction of death induced aberrations in multi-photon scanning microscopy using a deformable mirror” in “Journal of Microscopy”, vol. 206, 2002, p. 65-71), and P. M. Marsh et al. (“Practical implementation of adaptive optics in multi-photon microscopy” in “Optics Express”, vol. 11, 2003, p. 1123-1130).
These trial-and-error approaches can be used in thick, scattering samples such as e.g. brain tissue. However, these conventional procedures have the following disadvantages. Generally, the conventional procedures allow indirect measurements only. This means, that detecting of any wave-front distortion requires an illumination with a deformed illumination wavefront. Secondly, the iterative wave-front optimization requires the presence of a sufficiently bright fluorescence signal. Therefore, samples with sensitive fluorophores cannot be investigated due to photo bleaching. Furthermore, the iterative optimization is only feasible if the search space is sufficiently low dimensional, which limits the order to which distortion can be corrected. A further disadvantages is related to the high time consumption of the search algorithm using the deformable mirror.
M. A. A. Neil et al. (“Adaptive aberration correction in a two-photon-microscope” in “Journal of Microscopy”, vol. 200, 2000, p. 105-108) have proposed a direct measurement of the wave-front by evaluating the fluorescence emission of a specimen. The fluorescence measurement represents an essential disadvantage with regard to the investigation of sensitive samples.